SMART MODE:

NORMAL GENOMIC

• Kou WZ et al.
• Expression of Kin17 promotes the proliferation of hepatocellular carcinoma cells and
• Oncol Lett. 2014; 8: 1190-1194
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• Kin17 protein is ubiquitously expressed in mammals and is correlated with vital biological functions. However, little is known about the role of Kin17 in the proliferation of hepatocellular carcinoma cells. The aim of the present study was to investigate whether the upregulation of Kin17 can promote the growth of hepatocellular carcinoma cells. A series of assays was performed to study the effect of Kin17 in the proliferation of hepatocellular carcinoma cells in vitro and in vivo. The western blotting results revealed that Kin17 expression was increased in hepatocellular carcinoma tissues compared with that of the corresponding normal tissues. Moreover, ectopic upregulation of Kin17 expression promoted the growth of hepatocellular carcinoma cells in vitro and in vivo. These results indicated that Kin17 is involved in the tumorigenesis of hepatocellular carcinoma, and that Kin17 has the potential to serve as a therapeutic target for hepatocellular carcinoma.

• Tomicic MT, Reischmann P, Rasenberger B, Meise R, Kaina B, Christmann M
• Delayed c-Fos activation in human cells triggers XPF induction and an adaptive response to UVC-induced DNA damage and cytotoxicity.
• Cell Mol Life Sci. 2011; 68: 1785-98
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• The oncoprotein c-Fos has been commonly found differently expressed in cancer cells. Our previous work showed that mouse cells lacking the immediate-early gene c-fos are hypersensitive to ultraviolet (UVC) light. Here, we demonstrate that in human diploid fibroblasts UV-triggered induction of c-Fos protein is a delayed and long-lasting event. Sustained upregulation of c-Fos goes along with transcriptional stimulation of the NER gene xpf, which harbors an AP-1 binding site in the promoter. Data gained on c-Fos knockdown and c-Fos overexpressing human cells provide evidence that c-Fos/AP-1 stimulates upregulation of XPF, thereby increasing the cellular repair capacity protecting from UVC-induced DNA damage. When these cells are pre-exposed to a low non-toxic UVC dose and challenged with a subsequent high dose of UVC irradiation, they show accelerated repair of UVC-induced DNA adducts and reduced cell kill. The data indicate a protective role of c-Fos induction by triggering an adaptive response pathway.

• Zeng T et al.
• Up-regulation of kin17 is essential for proliferation of breast cancer.
• PLoS One. 2011; 6: 25343-25343
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• BACKGROUND: Kin17 is ubiquitously expressed at low levels in human tissue and participates in DNA replication, DNA repair and cell cycle control. Breast cancer cells are characterized by enabling replicative immortality and accumulated DNA damage. However, whether kin17 contributes to breast carcinogenesis remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we show for the first time that kin17 is an important molecule related to breast cancer. Our results show that kin17 expression was markedly increased in clinical breast tumors and was associated with tumor grade, Ki-67 expression, p53 mutation status and progesterone receptor expression, which were assessed in a clinicopathologic characteristics review. Knockdown of kin17 inhibited DNA replication and repair, blocked cell cycle progression and inhibited anchorage-independent growth, while increasing sensitivity to chemotherapy in breast cancer cells. Moreover, kin17 silencing decreased EGF-stimulated cell growth. Furthermore, overexpression of kin17 promoted DNA replication and cell proliferation in MCF-10A. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that up-regulation of kin17 is strongly associated with cellular proliferation, DNA replication, DNA damage response and breast cancer development. The increased level of kin17 was not only a consequence of immortalization but also associated with tumorigenesis. Therefore, kin17 could be a novel therapeutic target for inhibiting cell growth in breast cancer.

• Carlier L et al.
• Solution structure of the region 51-160 of human KIN17 reveals an atypical winged helix domain.
• Protein Sci. 2007; 16: 2750-5
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• Human KIN17 is a 45-kDa eukaryotic DNA- and RNA-binding protein that plays an important role in nuclear metabolism and in particular in the general response to genotoxics. Its amino acids sequence contains a zinc finger motif (residues 28-50) within a 30-kDa N-terminal region conserved from yeast to human, and a 15-kDa C-terminal tandem of SH3-like subdomains (residues 268-393) only found in higher eukaryotes. Here we report the solution structure of the region 51-160 of human KIN17. We show that this fragment folds into a three-alpha-helix bundle packed against a three-stranded beta-sheet. It belongs to the winged helix (WH) family. Structural comparison with analogous WH domains reveals that KIN17 WH module presents an additional and highly conserved 3(10)-helix. Moreover, KIN17 WH helix H3 is not positively charged as in classical DNA-binding WH domains. Thus, human KIN17 region 51-160 might rather be involved in protein-protein interaction through its conserved surface centered on the 3(10)-helix.

• le Maire A et al.
• A tandem of SH3-like domains participates in RNA binding in KIN17, a human protein activated in response to genotoxics.
• J Mol Biol. 2006; 364: 764-76
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• The human KIN17 protein is an essential nuclear protein conserved from yeast to human and expressed ubiquitously in mammals. Suppression of Rts2, the yeast equivalent of gene KIN17, renders the cells unviable, and silencing the human KIN17 gene slows cell growth dramatically. Moreover, the human gene KIN17 is up-regulated following exposure to ionizing radiations and UV light, depending on the integrity of the human global genome repair machinery. Its ectopic over-expression blocks S-phase progression by inhibiting DNA synthesis. The C-terminal region of human KIN17 is crucial for this anti-proliferation effect. Its high-resolution structure, presented here, reveals a tandem of SH3-like subdomains. This domain binds to ribonucleotide homopolymers with the same preferences as the whole protein. Analysis of its structure complexed with tungstate shows structural variability within the domain. The interaction with tungstate is mediated by several lysine residues located within a positively charged groove at the interface between the two subdomains. This groove could be the site of interaction with RNA, since mutagenesis of two of these highly conserved lysine residue weakens RNA binding.

• Biard DS, Despras E, Sarasin A, Angulo JF
• Development of new EBV-based vectors for stable expression of small interfering RNA to mimick human syndromes: application to NER gene silencing.
• Mol Cancer Res. 2005; 3: 519-29
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• We developed and characterized replicative small interfering RNA (siRNA) vectors for efficient, specific, and long-term gene silencing in human cells. We created stable XPA(KD) and XPC(KD) (knockdown) syngeneic cell lines to mimic human cancer-prone syndromes. We also silenced (HSA)KIN17. Several clones displaying undetectable protein levels of XPA, XPC, or (HSA)kin17 were grown for more than 300 days. This stability of gene silencing over several months of culture allows us to assess the specific involvement of these proteins in UVC sensitivity in syngeneic cells. Unlike XPA, (HSA)KIN17, and XPC gene silencing dramatically impeded HeLa cell growth for several weeks after transfection. As expected, XPA(KD) and XPC(KD) HeLa cells were highly UVC sensitive. They presented an impaired unscheduled DNA synthesis after UVC irradiation. Interestingly, XPC(KD) HeLa clones were more sensitive to UVC than their XPA(KD) or KIN17(KD) counterparts. Hygromycin B withdrawal led to the total disappearance of EBV vectors and the resumption of normal XPA or XPC protein levels. Whereas reverted XPA(KD) cells recovered a normal UVC sensitivity, XPC(KD) cells remained highly sensitive, suggestive of irreversible damage following long-term XPC silencing. Our results show that in HeLa cells, (HSA)kin17 participates indirectly in early events following UVC irradiation, and XPC deficiency strongly affects cell physiology and contributes to UVC sensitivity to a greater extent than does XPA. EBV-based siRNA vectors improve the interest of siRNA by permitting long-term gene silencing without the safety concerns inherent in viral-based siRNA vehicles.

• Hering TM et al.
• Characterization and chondrocyte differentiation stage-specific expression of KRAB zinc-finger protein gene ZNF470.
• Exp Cell Res. 2004; 299: 137-47
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• As part of a study to identify novel transcriptional regulators of chondrogenesis-related gene expression, we have cloned and characterized cDNA for zinc-finger protein 470 (ZNF470), the human ortholog of which encodes a 717 amino acid residue protein containing 17 Cys(2)His(2) zinc-finger domains, as well as KRAB-A and KRAB-B motifs. The cDNA library used to isolate the initial ZNF470 clone was prepared from human bone marrow-derived mesenchymal progenitor cells at an intermediate stage of chondrogenic differentiation. We have determined the intron-exon structure of the human ZNF470 gene, which has been mapped to a zinc-finger cluster in a known imprinted region of human chromosome 19q13.4. ZNF470 is expressed at high levels in human testis and is expressed at low or undetectible levels in other adult tissues. Human ZNF470 expressed in mammalian cells as an EGFP fusion protein localizes predominantly to the nucleus, consistent with a role in transcriptional regulation. ZNF470, analyzed by quantitative real time PCR, was transiently expressed before the maximal expression of COL2A1 during chondrogenic differentiation in vitro. We have also characterized the bovine ortholog of human ZNF470, which encodes a 508 amino acid residue protein having 10 zinc-finger domains. A bovine ZNF470 cDNA clone was used to examine expression of ZNF470 in bovine articular chondrocytes treated with retinoic acid to stimulate dedifferentiation. Bovine ZNF470 expression was undetectable in freshly isolated bovine articular chondrocytes, but was dramatically upregulated in dedifferentiated retinoic acid-treated chondrocytes. These results, in two model systems, suggest a possible role for ZNF470 in the regulation of chondrogenesis-specific gene expression.

• Gianfrancesco F, Esposito T, Casu G, Maninchedda G, Roberto R, Pirastu M
• Emergence of Talanin protein associated with human uric acid nephrolithiasis in the Hominidae lineage.
• Gene. 2004; 339: 131-8
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• Recently, we identified a susceptibility locus for human uric acid nephrolithiasis (UAN) on 10q21-q22 and demonstrated that a novel gene (ZNF365) included in this region produces through alternative splicing several transcripts coding for four protein isoforms. Mutation analysis showed that one of them (Talanin) is associated with UAN. We examined the evolutionary conservation of ZNF365 gene through a comparative genomic approach. Searching for mouse homologs of ZNF365 transcripts, we identified a highly conserved mouse ortholog of ZNF365A transcript, expressed specifically in brain. We did not found a mouse homolog for ZNF365D transcript encoding the Talanin protein, even if we were able to identify the corresponding genomic region in mouse and rat not yet organized in canonical gene structure suggesting that ZNF365D was originated after the branching of hominoid from rodent lineage. In mouse and in most mammals, a functional uricase degrades the uric acid to allantoin, but uricase activity was lost during the Miocene epoch in hominoids. Searching for the presence of Talanin in Primates, we found a canonical intron-exon structure with several stop codons preventing protein production in Old World and New World monkeys. In humans, we observe expression and we have evidence that ZNF365D transcript produces a functional protein. It seems therefore that ZNF365D transcript emerged during primate evolution from a noncoding genomic sequence that evolved in a standard gene structure and assumed its role in parallel with the disappearance of uricase, probably against a disadvantageous excessive hyperuricemia.

• Wang Z, Peters B, Klussmann S, Bender H, Herb A, Krieglstein K
• Gene structure and evolution of Tieg3, a new member of the Tieg family of proteins.
• Gene. 2004; 325: 25-34
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• TGF beta-inducible immediate early gene, Tieg, belongs to the superfamily of Sp1-like transcription factors containing three C(2)H(2)-zinc finger DNA binding motifs close to the C-terminus. So far, Tieg1 and Tieg2 have been identified in human and mouse. We identified Tieg3, a new member of the Tieg protein family by screening a mouse cDNA library. Tieg3 has almost all the known features of the Tieg protein family: it shares a highly conserved C(2)H(2) zinc finger DNA binding domain and is 96% identical to Tieg2 and 86% to Tieg1, respectively. In addition, the three repression domains at the N-terminus, R1, R2 and R3 are conserved in all the Tiegs. Similar to Tieg1 and Tieg2, Tieg3 mRNA is up-regulated in response to TGF beta 1 treatment and can perform the Sp1 sites mediated repression of transcription. A 4 kilobase (kb) long transcript of mouse Tieg3 can be detected using Northern-blot analysis. The gene of mouse Tieg3 contains four exons. Due to the amino acid sequence similarity, mouse Tieg2 is regarded as an orthologue of human Tieg2. However, the mouse Tieg3 gene is localized in a conserved segment on mouse chromosome 12 corresponding to human Tieg2 on chromosome 2 with the same gene order. An interesting explanation for this apparent contradiction might be a homologous recombination leading to loci exchange between the mouse Tieg3 and Tieg2.

• Masson C et al.
• Global genome repair is required to activate KIN17, a UVC-responsive gene involved in DNA replication.
• Proc Natl Acad Sci U S A. 2003; 100: 616-21
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• UV light provokes DNA lesions that interfere with replication and transcription. These lesions may compromise cell viability and usually are removed by nucleotide excision repair (NER). In humans, inactivation of NER is associated with three rare autosomal recessive inherited disorders: xeroderma pigmentosum (XP), Cockayne syndrome, and trichothiodystrophy. The NER earliest step is lesion recognition by a complex formed by XPC and HHR23B proteins. In a subsequent step, XPA protein becomes associated to the repair complex. Here we investigate whether XPA and XPC proteins, involved in global genome repair, may contribute to a signal transduction pathway regulating the response to UVC-induced lesions. We monitored the expression of several UVC-induced genes in cells deficient in either a transduction pathway or mutated on an NER gene. Expression of the KIN17 gene is induced after UVC irradiation independently of p53 and of activating transcription factor 2. However, in human cells derived from XPA or XPC patients the UVC-induced accumulation of KIN17 RNA and protein is abolished. Our results indicate that the presence of functional XPA and XPC proteins is essential for the up-regulation of the KIN17 gene after UVC irradiation. They also show that the integrity of global genome repair is required to trigger KIN17 gene expression and probably other UVC-responsive genes.

• Spassov DS, Jurecic R
• Mouse Pum1 and Pum2 genes, members of the Pumilio family of RNA-binding proteins, show differential expression in fetal and adult hematopoietic stem cells and progenitors.
• Blood Cells Mol Dis. 2003; 30: 55-69
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• Self-renewal is the common functional property of all types of stem cells and is thought to be regulated by unknown conserved intrinsic and extrinsic molecular mechanisms. Recently, an evolutionarily conserved Pumilio family of RNA-binding proteins that regulate asymmetric cell division was found to be essential for stem cell maintenance and self-renewal in Drosophila and Caenorhabditis elegans. Based on conserved function in invertebrates and lower vertebrates it was recently proposed that an ancestral function of Pumilio proteins is to support proliferation and self-renewal of stem cells. This raises an interesting possibility that Pumilio could be part of evolutionarily conserved intrinsic molecular mechanism that regulates self-renewal of mammalian stem cells. Here we describe cloning and comparative sequence analysis of Pum1 and Pum2 genes, mouse members of the Pumilio family, and for the first time demonstrate expression of Pumilio genes in mammalian hematopoietic stem cells (HSC). Pum1 and Pum2 share 51 and 55% overall similarity with the fly Pum, whereas their RNA-binding domains show a very high degree of evolutionary conservation (86-88% homology). Both genes are expressed in a variety of tissues suggesting that they have widespread function. During blood cell development Pum1 and Pum2 exhibit differential expression in cell populations enriched for HSC and progenitors. Both genes are highly transcribed in populations of adult HSC (Rho-123(low)Sca-1(+)c-kit(+)Lin(-) cells). In a more heterogeneous population of HSC (Lin(-)Sca-1(+)) and in progenitors (Lin(-)Sca-1(-) cells) Pum1 is not transcribed, whereas Pum2 expression is significantly down-regulated. Ongoing in vitro and in vivo functional analysis of mouse Pumilio genes will help to elucidate the biological role of mammalian Pumilio genes and determine whether they play any role in maintenance of mammalian stem cells, such as HSC.

• Luo X, Huang Y, Sheikh MS
• Cloning and characterization of a novel gene PDRG that is differentially regulated by p53 and ultraviolet radiation.
• Oncogene. 2003; 22: 7247-57
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• We report the cloning and characterization of a novel p53 and DNA damage-regulated gene (PDRG). The human and mouse PDRG sequences are highly homologous and contain open reading frames of 133 amino acids each with molecular masses of 15.5 and 15.3 kDa, respectively. PDRG codes for a novel protein that does not show similarity to any known protein in the databases. Human PDRG is predominantly expressed in normal testis and exhibits reduced but detectable expression in other organs. GFP-tagged PDRG was predominantly detected as aggregates that appeared to reside in a distinct subcellular compartment. PDRG mRNA was upregulated by ultraviolet radiation (UV) but downregulated by tumor suppressor p53. UV is known to transcriptionally upregulate the expression of certain genes by activating the transcription factor Oct-1, while p53 has been reported to suppress transcription of certain genes by directly binding to a novel head-to-tail response element. Cloning and sequence analysis of PDRG promoter revealed the presence of Oct-1-binding element and a putative head-to-tail-type p53-binding site. Indeed, UV as well as exogenous Oct-1 independently increased PDRG promoter activity, suggesting that UV could mediate PDRG upregulation via Oct-1. Exogenous wild-type p53 was found to downregulate the PDRG promoter activity indicating that wild-type p53 transcriptionally suppresses the expression of PDRG and may mediate its effect via the putative head-to-tail response element. Furthermore, stable expression of exogenous PDRG was found to decrease the clonogenic survival after UV irradiation, which highlights the significance of PDRG in facilitating UV-induced killing.

• Carlone DL, Hart SR, Ladd PD, Skalnik DG
• Cloning and characterization of the gene encoding the mouse homologue of CpG binding protein.
• Gene. 2002; 295: 71-7
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• Human CpG binding protein (CGBP) is a ubiquitously-expressed transcriptional activator that binds specifically to unmethylated CpG motifs. Several protein domains have been identified within CGBP including two plant homeodomains (PHD), acidic and basic regions, a coiled-coil domain, as well as a CXXC DNA-binding domain. The global function of CGBP remains unclear, although failure to express CGBP results in embryonic lethality in mice. This study reports the identification and characterization of the murine CGBP gene locus. A 2509 bp murine CGBP cDNA was cloned and nucleotide sequence determined. Comparison of the mouse and human CGBP sequences revealed 86% identity at the nucleotide level and 96% identity at the amino acid level. Examination of the deduced translation product revealed that the PHD, CXXC, coiled-coil, and basic domains are identical between mouse and human, while the acidic region exhibits approximately 90% identity with its human counterpart. A single murine CGBP transcript of approximately 2.6 kb was detected in a wide variety of adult tissues as well as embryonic stem cells. Analysis of the mouse gene locus revealed a relatively small gene spanning approximately 5 kb and comprised of 15 exons. Examination of the human CGBP gene showed a similar size and structure with identical intronic splice sites. In contrast to the human CGBP gene, which is located 800 bp upstream of the MBD1 gene, analysis of the murine CGBP gene locus failed to detect the murine MBD1 gene within several kilobases of the CGBP coding region.

• Squillace RM, Chenault DM, Wang EH
• Inhibition of muscle differentiation by the novel muscleblind-related protein CHCR.
• Dev Biol. 2002; 250: 218-30
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• Growth factor withdrawal from proliferating myoblasts induces the expression of muscle-specific genes essential for myogenesis. By suppression subtractive hybridization (SSH), we have cloned a novel human cDNA that encodes a Cys3His zinc finger protein named CHCR (Cys3His CCG1-Required). CHCR is related to Muscleblind (Mbl), a Drosophila melanogaster protein required for terminal muscle differentiation. It also displays sequence similarity to EXP/MBNL, a human Mbl protein that interacts with CUG expansions associated with the degenerative muscular disease, myotonic dystrophy (DM1). This relationship with EXP/MBNL and Mbl suggests that CHCR also functions during muscle differentiation. We have found that CHCR mRNA and protein levels decrease upon differentiation of mouse myoblast cells. Constitutive expression of CHCR in C2C12 cells inhibits the induction of sarcomeric myosin heavy chain (MyHC) upon serum deprivation. Induction of myogenin, an earlier marker of muscle differentiation, is inhibited to a lesser extent, while expression of the cell cycle inhibitor, p21, remains unaffected. Loss of CHCR function by morpholino antisense oligonucleotide treatment accelerates MyHC induction during differentiation of myoblast cells. These complementary gain- and loss-of-function results suggest that CHCR is an inhibitor of myogenesis. CHCR represents the first muscleblind-related protein that antagonizes, instead of promotes, muscle differentiation.

• Tune CE, Pilon M, Saiki Y, Dosch HM
• Sustained expression of the novel EBV-induced zinc finger gene, ZNFEB, is critical for the transition of B lymphocyte activation to oncogenic growth transformation.
• J Immunol. 2002; 168: 680-8
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• EBV is a human tumor virus that infects and establishes latency in the majority of humans worldwide. In vitro, EBV growth transforms primary B lymphocytes into lymphoblastoid cell lines with high efficiency. We have used cDNA subtraction cloning to identify cellular target genes required for growth transformation and identified a new C(2)H(2) (Kruppel-type) zinc finger gene, ZNF(EB), that is trans-activated early following EBV infection. In this study, we characterize ZNF(EB), including its intronless locus, and human and mouse protein variants. The gene is transiently expressed during normal lymphocyte activation, and its expression is sustained in EBV-positive but not EBV-negative B cell lines. There is limited expression in nonhemopoietic tissues. Its critical role in the growth transformation of B lineage cells is indicated by the abrogation of transformation with antisense strategies. ZNF(EB) maps to chromosome 18q12, a region with mutations in numerous, predominantly hemopoietic malignancies.

• Qi H et al.
• AIbZIP, a novel bZIP gene located on chromosome 1q21.3 that is highly expressed in prostate tumors and of which the expression is up-regulated by androgens in LNCaP human prostate cancer cells.
• Cancer Res. 2002; 62: 721-33
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• Androgens play an important role in the development and physiology of the normal prostate as well as in prostate cancer cell proliferation. Comparison of the mRNA expression profiles of control and R1881-treated cultures of LNCaP human prostate cancer cells using cDNA subtraction led to the identification of a novel transcription factor that we named Androgen-Induced bZIP (AIbZIP) protein. AIbZIP is a 395 aa protein with homology to cyclic AMP-responsive element binding protein/activating transcription factor transcription factors. It contains an NH(2)-terminal activation domain, a central bZIP domain, and a COOH-terminal transmembrane domain. The AIbZIP gene is localized on chromosome 1q21.3 and consists of 10 exons. A major 1.7-kb transcript was detected exclusively in the prostate as well as in breast and prostate cancer cell lines. Androgens up-regulate AIbZIP mRNA and protein levels in a dose-dependent manner. The kinetics of AIbZIP mRNA up-regulation and the results of experiments with cycloheximide suggest that AIbZIP may be a delayed response gene. Immunoreactive AIbZIP protein was primarily detected in the cytoplasm of prostatic luminal epithelial cells. Similarly, full-length AIbZIP-green fluorescent protein fusion proteins were localized in the cytoplasm of LNCaP cells, whereas a truncated form of AIbZIP lacking the putative transmembrane domain was exclusively nuclear. Examination of AIbZIP protein and mRNA expression in a series of transurethral resection of the prostate and needle biopsy specimens indicated that AIbZIP is expressed at higher levels in cancerous prostate cells compared with noncancerous prostate cells. The highly tissue-specific expression profile, androgen regulation, chromosomal localization, and expression profile of AIbZIP in prostate tumors suggest that AIbZIP may play an important role in prostate cancer and in androgen receptor signaling in prostate cells. Future studies will confirm a possible relationship between AIbZIP and prostate cancer.

• Masson C, Menaa F, Pinon-Lataillade G, Frobert Y, Radicella JP, Angulo JF
• Identification of KIN (KIN17), a human gene encoding a nuclear DNA-binding protein, as a novel component of the TP53-independent response to ionizing radiation.
• Radiat Res. 2001; 156: 535-44
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• Ionizing radiation elicits a genetic response in human cells that allows cell survival. The human KIN (also known as KIN17) gene encodes a 45-kDa nuclear DNA-binding protein that participates in the response to UVC radiation and is immunologically related to the bacterial RecA protein. We report for the first time that ionizing radiation and bleomycin, a radiomimetic drug, which produce single- and double-strand breaks, increased expression of KIN in human cells established from tumors, including MeWo melanoma, MCF7 breast adenocarcinoma, and ATM+ GM3657 lymphoblast cells. KIN expression increased rapidly in a dose-dependent manner after irradiation. Under the same conditions, several genes controlled by TP53 were induced with kinetics similar to that of KIN. Using the CDKN1A gene as a marker of TP53 responsiveness, we analyzed the up-regulation of KIN and showed that is independent of the status of TP53 and ATM. In contrast, the presence of a dominant mutant for activating transcription factor 2 (ATF2) completely abolished the up-regulation of KIN. Our results suggest a role for ATF2 in the TP53-independent increase in KIN expression after gamma irradiation.

• Misawa H, Yamaguchi M
• Molecular cloning and sequencing of the cDNA coding for a novel regucalcin gene promoter region-related protein in rat, mouse and human liver.
• Int J Mol Med. 2001; 8: 513-20
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• The molecular cloning and sequencing of the cDNA coding for a novel regucalcin gene promoter region-related protein (RGPR) was investigated using rat, mouse and human liver cDNA library with a yeast one-hybrid system and a rapid amplification of cDNA ends (RACE) method. The clone coding an unknown protein was isolated, and a novel protein was identified. This protein was termed as RGPR-p117. RGPR-p117 in rat, mouse and human liver consisted of 1058, 1051 and 1060 amino acid residues with calculated molecular mass of 117, 115 and 117 kDa and estimated pI of 5.69, 5.70 and 5.71, respectively. The homologies of amino acids among rat, mouse and human RGPR-p117 were at least 70%. RGPR-p117 had a leucine zipper motif. The expression of RGPR-p117 mRNA was found in the liver, kidney, heart, spleen, and brain of rats. The database search of the human RGPR-p117 showed that its gene consisted of at least 26 exons spanning approximately 4.1 kbp and localized on human chromosome 1q25.2. Furthermore, we found a cDNA clone which was highly identical to a front half part of the human RGPR-p117 cDNA, using the BLAST search of human RGPR-p117. This cDNA clone was a splicing variant of human RGPR-p117, which derived from human placental choriocarcinoma. Our study demonstrates that a novel gene coding RGPR-p117 is present in rat, mouse and human.

• Hellborg F et al.
• Human wig-1, a p53 target gene that encodes a growth inhibitory zinc finger protein.
• Oncogene. 2001; 20: 5466-74
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• We previously identified a novel p53-induced mouse gene, wig-1, that encodes a 290 amino acid zinc finger protein (Varmeh-Ziaie et al., 1997). Here we have identified and characterized the human homolog of mouse wig-1. The human wig-1 protein is 87% identical to the mouse protein and contains three zinc finger domains and a putative nuclear localization signal. Human wig-1 mRNA and protein is induced following activation of wild type p53 expression in our BL41-ts p53 Burkitt lymphoma cells. Wig-1 is also induced in MCF7 cells following treatment with the DNA-damaging agent mitomycin C. Northern blotting detected low levels of wig-1 mRNA in normal human tissues. Fluorescence in situ hybridization mapped wig-1 to human chromosome 3q26.3-27. FLAG-tagged human wig-1 localizes to the nucleus. Ectopic overexpression of human wig-1 inhibits tumor cell growth in a colony formation assay. These results suggest that human wig-1 has a role in the p53-dependent growth regulatory pathway.

• Ostvold AC, Norum JH, Mathiesen S, Wanvik B, Sefland I, Grundt K
• Molecular cloning of a mammalian nuclear phosphoprotein NUCKS, which serves as a substrate for Cdk1 in vivo.
• Eur J Biochem. 2001; 268: 2430-40
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• We have isolated and characterized a cDNA encoding a mammalian nuclear phosphoprotein NUCKS, previously designated P1. Molecular analyses of several overlapping and full-length cDNAs from HeLa cells and rat brain revealed a protein with an apparent molecular mass of 27 kDa in both species. The deduced amino-acid sequences are highly conserved between human and rodents, but show no homology with primary structures in protein databases or with translated sequences of cDNAs in cDNA databanks. Although the protein has some features in common with the high mobility group proteins HMGI/Y, attempts to find a putative protein family by database query using both sequence alignment methods and amino-acid composition have failed. Northern blot analyses revealed that human and rat tissues contain three NUCKS transcripts varying in size from 1.5 to 6.5 kb. All human and rat tissues express the gene, but the level of transcripts varies among different tissues. Circular dichroism analysis and secondary structure predictions based on the amino-acid sequence indicate a low level of alpha helical content and substantial amounts of beta turn structures. The protein is phosphorylated in all phases of the cell cycle and exhibits mitosis-specific phosphorylation of threonine residues. Phosphopeptide mapping and back-phosphorylation experiments employing NUCKS from HeLa interphase and metaphase cells show that the protein is phosphorylated by Cdk1 during mitosis of the cell cycle.

• Seki N, Ueki N, Yano K, Saito T, Masuho Y, Muramatsu M
• cDNA cloning of a novel human gene NAKAP95, neighbor of A-kinase anchoring protein 95 (AKAP95) on chromosome 19p13.11-p13.12 region.
• J Hum Genet. 2000; 45: 31-7
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• A-kinase anchoring protein 95 (AKAP95) is a nuclear protein which binds to the regulatory subunit (RII) of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and to DNA. A novel nuclear human gene which shares sequence homology with the human AKAP95 gene was identified by a nuclear transportation trap method. By polymerase chain reaction (PCR)-based analysis with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid panel, the gene was mapped to the chromosome 19p13.11-p13.12 region between markers WI-4669 and CHLC.GATA27C12. Furthermore, alignment with genomic sequences revealed that the gene and human AKAP95 resided tandemly only approximately 250 bp apart from each other. We designated this gene as neighbor of AKAP95 (NAKAP95). The exon-intron structure of NAKAP95 and AKAP95 was conserved, indicating that they may have evolved by gene duplication. The predicted protein product of the NAKAP95 gene consists of 646 amino acid residues, and NAKAP95 and AKAP95 had an overall 40% similarity, both having a potential nuclear localizing signal and two C2H2 type zinc finger motifs. The putative RII binding motif in AKAP95 was not conserved in NAKAP95. A reverse transcription coupled (RT)-PCR experiment revealed that the NAKAP95 gene was transcribed ubiquitously in various human tissues.

• Saeki N, Kuwahara Y, Sasaki H, Satoh H, Shiroishi T
• Gasdermin (Gsdm) localizing to mouse Chromosome 11 is predominantly expressed in upper gastrointestinal tract but significantly suppressed in human gastric cancer cells.
• Mamm Genome. 2000; 11: 718-24
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• Amplification of proto-oncogenes associated with their over-expression is one of the critical carcinogenic events identified in human cancer cells. In many cases of human gastric cancer, a proto-oncogene ERBB-2 is co-amplified with CAB1 genes physically linked to ERBB-2, and both genes are over-expressed. The amplified region containing ERBB-2 and CAB1 was named 17q12 amplicon from its chromosomal location. The syntenic region corresponding to the 17q12 amplicon is well conserved in mouse. In this study we isolated and characterized a novel mouse gene that locates telomeric to the mouse syntenic region. Northern blot analysis using the mouse cDNA and a cloned partial cDNA of human homolog disclosed a unique expression pattern of the genes. They are expressed predominantly in the gastrointestinal (GI) tract and in the skin at a lower level. Moreover, in the GI tract, the expression is highly restricted to the esophagus and stomach. Thus, we named the mouse gene Gasdermin (Gsdm). This is the first report of a mammalian gene whose expression is restricted to both upper GI tract and skin. Interestingly, in spite of its expression in normal stomach, no transcript was detected by Northern blot analysis in human gastric cancer cells. These data suggest that the loss of the expression of the human homolog is required for the carcinogenesis of gastric tissue and that the gene has an activity adverse to malignant transformation of cells.

• Bridger JM, Boyle S, Kill IR, Bickmore WA
• Re-modelling of nuclear architecture in quiescent and senescent human fibroblasts.
• Curr Biol. 2000; 10: 149-52
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• Spatial organisation of the genome within the nucleus can play a role in maintaining the expressed or silent state of some genes [1]. There are distinct addresses for specific chromosomes, which have different functional characteristics, within the nuclei of dividing populations of human cells [2]. Here, we demonstrate that this level of nuclear architecture is altered in cells that have become either quiescent or senescent. Upon cell cycle exit, a gene-poor human chromosome moves from a location at the nuclear periphery to a more internal site in the nucleus, and changes its associations with nuclear substructures. The chromosome moves back toward the edge of the nucleus at a distinctive time after re-entry into the cell cycle. There is a 2-4 hour period at the beginning of G1 when the spatial organisation of these human chromosomes is established. Lastly, these experiments provide evidence that temporal control of DNA replication can be independent of spatial chromosome organisation. We conclude that the sub-nuclear organisation of chromosomes in quiescent or senescent mammalian somatic cells is fundamentally different from that in proliferating cells and that the spatial organisation of the genome is plastic.

• Bolivar J, Diaz I, Iglesias C, Valdivia MM
• Molecular cloning of a zinc finger autoantigen transiently associated with interphase nucleolus and mitotic centromeres and midbodies. Orthologous proteins with nine CXXC motifs highly conserved from nematodes to humans.
• J Biol Chem. 1999; 274: 36456-64
• Display abstract

• We have cloned a novel human autoimmune antigen in a patient suffering from rheumatoid arthritis with high levels of antibodies to the nucleolus organizer regions. Initially the human autoimmune serum was used to select a cDNA of 317 amino acids from a hamster expression library. Using the hamster DNA as a probe, we isolated the human homologous cDNA of 320 amino acids. Human and hamster polypeptides share a 95% amino acid homology. The deduced 36-kDa protein contains a putative amino-terminal NLS signal, nine cysteine-X-X-cysteine motifs highly conserved, and a carboxyl-terminal poly acidic region. Several homologous expressed sequence tags have been identified in data bases suggesting that orthologous proteins are present throughout evolution from worms to humans. A Drosophila expressed sequence tag was further completely sequenced for a full-length protein with 60% amino acid identity to the human homologue. Northern blot analysis revealed that this novel protein is widely distributed in human tissues with significantly higher expression levels in heart and skeletal muscle. Specific antibodies to the recombinant protein and transfection experiments demonstrated by immunofluorescence the localization of the protein predominantly but not exclusively to the nucleolus of interphase mammalian cells. In actinomycin D-treated cells the protein remains associated with the nucleolus but is not segregated, like other ribosomal factors such as upstream binding factor. In mitosis the protein was found to be associated with centromeres and concentrated at the midbody in cytokinesis. Transient distribution of this evolutionarily conserved zinc finger nucleolar autoantigen to the mitotic centromeres may provide the means for several aspects of cell cycle control and transcriptional regulation.

• Holmes DI, Wahab NA, Mason RM
• Cloning and characterization of ZNF236, a glucose-regulated Kruppel-like zinc-finger gene mapping to human chromosome 18q22-q23.
• Genomics. 1999; 60: 105-9
• Display abstract

• We report the cDNA cloning and characterization of ZNF236, a novel Kruppel-like zinc-finger gene initially identified by its glucose-regulated expression in human mesangial cells using mRNA differential display. Using the differential display fragment as a probe, we screened a human fetal kidney cDNA library and isolated several clones representing two differently spliced mRNA transcripts, designated ZNF236a and -b. Both transcripts were identical apart from the presence of an additional exon in ZNF236a that truncates the open reading frame. RT-PCR analysis confirmed the expression of both transcripts to be upregulated in human mesangial cells in response to elevated levels of d-glucose. ZNF236a and -b cDNAs encode polypeptides of 174 and 204 kDa, containing 25 and 30 C(2)H(2) zinc-finger motifs, respectively. Northern blot analysis showed that ZNF236 is ubiquitously expressed in all human tissues tested. Expression levels were highest in skeletal muscle and brain, intermediate in heart, pancreas, and placenta, and lowest in kidney, liver, and lung. Southern zoo blot analysis indicated that ZNF236 is conserved in the genomes of all mammalian species tested, but not in yeast. The mapping of ZNF236 to human chromosome 18q22-q23, close to the IDDM6 locus, coupled with the glucose-regulated expression of the gene in human mesangial cells, suggests that ZNF236 may be a candidate gene for diabetic nephropathy.

• Inada H, Naka M, Tanaka T, Davey GE, Heizmann CW
• Human S100A11 exhibits differential steady-state RNA levels in various tissues and a distinct subcellular localization.
• Biochem Biophys Res Commun. 1999; 263: 135-8
• Display abstract

• In order to analyze the steady-state RNA levels of S100A11 in different tissues, a cDNA fragment of human S100A11 was isolated from a cDNA library. The obtained fragment was labeled and hybridized to RNA isolated from various tissues. The Northern blot analysis revealed that S100A11 RNA levels varied from high in placenta, through intermediate in heart, lung, kidney, and most muscle samples, to barely detectable in brain. An efficient purification method for recombinant S100A11 yielding high quantities was developed. Furthermore, to examine the subcellular localization of this protein, the human polypeptide S100A11 antibodies were raised in rabbit. S100A11 was found to have a localization distinct from other S100 proteins examined, and is mostly localized in the nucleus, with slight variations among different glioblastoma cell types.

• Ohbayashi T et al.
• Isolation of cDNA, chromosome mapping, and expression of the human TBP-like protein.
• Biochem Biophys Res Commun. 1999; 255: 137-42
• Display abstract

• TBP is an essential factor for eukaryotic transcription. In this study, we identified a human cDNA encoding 21-kDa TBP-like protein (TLP). The TLP ORF, carrying 186 amino acids, covered the entire 180 amino acids of the C-terminal conserved domain of human TBP with 39% identity and 76% similarity. FISH determined that human tlp gene was located at chromosome 6 region q22.1-22.3. Northern blot analysis demonstrated that TLP mRNAs were expressed in various human tissues ubiquitously. We found that the TLP proteins exist in multiple mammalian cells and chicken cells. Although the Drosophila TBP-related factor (TRF) is a neurogenesis-related transcription factor, expression of TLP was nearly constant throughout the neural differentiation of P19 cells. Unlike TRF, TLP did not bind to the TATA-box nor direct transcription initiation in vitro. Similarity between TRF and TLP was considerably lower (35 in alignment score) than that between Drosophila TBP and human TBP (88 in alignment score). Multiple amino acids critical for the TBP function were deleted or substituted in TLP. We suggest that TLP is not a bona fide vertebrate counterpart nor a direct descendant of TRF.

• Inoue S et al.
• Molecular cloning of rat efp: expression and regulation in primary osteoblasts.
• Biochem Biophys Res Commun. 1999; 261: 412-8
• Display abstract

• We have previously identified an estrogen-responsive gene, efp (estrogen-responsive finger protein), by genomic binding-site cloning method. Here, we isolated a rat homologue of efp cDNA that encodes an open reading frame of 644 amino acids sharing high homology with human efp (69% identity at the protein level) and mouse efp (80% identity at the protein level). The efp protein has a RING finger, a variant type of zinc finger motif, B1 box and B2 box, each having a pair of zinc fingers, and coiled-coil domain, belonging to the RING finger-B box-Coiled Coil (RBCC) family. Several members of RBCC family including efp have characteristic C-terminal domain, forming a subfamily. Next, we detected efp mRNA in primary osteoblasts, one of estrogen target cells, derived from the calvariae of rat fetus. An anti-efp antibody revealed the efp protein is expressed and regulated by estrogen in the primary osteoblasts. Interestingly, the efp protein in primary osteoblasts is down-regulated by 1alpha,25-dihydroxyvitamin D(3) treatment that promotes the differentiation of the cells, whereas it is up-regulated by TGF-beta1 treatment that inhibits the differentiation of the cells. These findings suggest the possible involvement of the efp in the differentiation of osteoblastic cells.

• Meagher MJ et al.
• Identification of ZFR, an ancient and highly conserved murine chromosome-associated zinc finger protein.
• Gene. 1999; 228: 197-211
• Display abstract

• In a screen for RNA binding proteins expressed during murine spermatogenesis, we cloned a novel, ancient zinc finger protein possessing a region common to a small class of RNA binding proteins. Zfr (zinc finger RNA binding) encodes a protein of 1052 amino acids with three widely spaced Cys2His2 zinc fingers. Outside of the zinc fingers, ZFR shares a region that is highly conserved between several RNA binding proteins containing copies of the double-stranded RNA binding motif. By northern blotting, Zfr is expressed at highest levels within the testis, ovary and brain. Immunohistochemistry and confocal microscopy were used to show that ZFR is highly expressed during meiosis I in males and females and is chromosome associated. Zfr is also expressed in Sertoli cells in the testis and granulosa cells in the ovary where it is localized to the nucleus. Using fluorescent in situ hybridization we mapped Zfr to chromosome 15 region A. ZFR appears to be an ancient protein, as apparent homologs exist in invertebrates (D. melanogaster) nematodes (C. elegans) and humans (H. sapiens).

• Tu Y, Wu C
• Cloning, expression and characterization of a novel human Ras-related protein that is regulated by glucocorticoid hormone.
• Biochim Biophys Acta. 1999; 1489: 452-6
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• Ras proteins are a family of guanine nucleotide (GDP and GTP)-binding proteins that play central roles in essential signal transduction pathways. We have isolated in a yeast two-hybrid screen a human cDNA encoding a new protein that is highly homologous (98% identical at the protein level) to mouse DexRas1, a member of the Ras superfamily. The human DexRas1 is expressed in a variety of tissues including heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas, with the strongest expression in the heart. Using human fibrosarcoma HT-1080 cells as a model system, we show that the expression of human DexRas1 is stimulated by dexamethasone, suggesting a role of human DexRas1 in dexamethasone-induced alterations in cell morphology, growth and cell-extracellular matrix interactions.

• Buaas FW, Lee K, Edelhoff S, Disteche C, Braun RE
• Cloning and characterization of the mouse interleukin enhancer binding factor 3 (Ilf3) homolog in a screen for RNA binding proteins.
• Mamm Genome. 1999; 10: 451-6
• Display abstract

• In a screen for RNA-binding proteins expressed during murine spermatogenesis, we have identified a cDNA that encodes a protein of 911 amino acids that contains two copies of the double-stranded RNA-binding motif and has 80% identity with human Interleukin Enhancer Binding Factor 3 (ILF3). Linkage and cytogenetic analyses localized the Ilf3 cDNA to a portion of mouse Chr 9, which shows conserved synteny with a region of human Chr 19 where the human ILF3 gene had been previously localized, supporting that we had cloned the murine homolog of ILF3. Northern analysis indicated the Ilf3 gene is ubiquitously expressed in mouse adult tissues with high levels of expression in the brain, thymus, testis, and ovary. Polyclonal antibodies detected multiple protein species in a subset of the tissues expressing Ilf3 RNA. Immunoreactive species are present at high levels in the thymus, testis, ovary, and the spleen to a lesser extent. The high degree of sequence similarity between the mouse ILF3 protein and other dsRNA binding motif-containing proteins suggests a role in RNA metabolism, while the differential expression indicates the mouse ILF3 protein predominantly functions in tissues containing developing lymphocyte and germ cells.

• Houle JF, Friedberg EC
• The Drosophila ortholog of the human XPG gene.
• Gene. 1999; 234: 353-60
• Display abstract

• Xeroderma pigmentosum complementation group G (XPG) protein is a junction-specific endonuclease which is indispensable for nucleotide excision repair (NER) of DNA in eukaryotes. Recent studies have hinted at a second, essential function for the XPG protein in higher eukaryotes. We undertook a comparison of the amino acid sequences of multiple XPG orthologs to determine if a motif or domain could be identified that is conserved uniquely in higher eukaryotes. A search of current databases allowed us to retrieve complete amino acid sequences for the human, mouse and Xenopus XPG proteins, and for two yeast orthologs. We also identified an incomplete Drosophila open reading frame (ORF) that was a good candidate for the XPG protein. We cloned a complete Drosophila cDNA for this ORF and examination of the primary amino acid sequence suggests that this cDNA encodes the Drosophila ortholog of XPG. A comparison of all six orthologous polypeptides reveals the presence of two previously unidentified conserved domains. One of these is unique to all four higher eukaryotic sequences. Conceivably this domain evolved to support the essential function of XPG protein.

• Miyazaki S, Rasmussen S, Imatani A, Diella F, Sullivan DT, Callahan R
• Characterization of the Drosophila ortholog of mouse eIF-3p48/INT-6.
• Gene. 1999; 233: 241-7
• Display abstract

• The mouse mammary tumor virus (MMTV) has been shown to integrate frequently into INT-6 in MMTV-induced mouse mammary tumors. The INT6 gene has been highly conserved through evolution and has recently been shown to encode the p48 component of the eucaryotic translation initiation factor 3 (eIF-3) complex. We report here the isolation of the Drosophila eIF-3p48/INT-6. The gene comprises three exons within 1.8kb of genomic DNA located at cytogenetic position 73C2 in the Drosophila genome. The 1.5kb eIF-3p48/INT-6 RNA species encodes a protein composed of 364 amino-acid residues whose sequence is 71% similar to that of the mouse/human eIF-3/INT-6 amino-acid sequence. eIF-3p48/INT-6 RNA is expressed throughout development in Drosophila and the encoded protein is associated with the microsomal subcellular fraction.

• Nishio H, Suda T, Sawada K, Miyamoto T, Koike T, Yamaguchi Y
• Molecular cloning of cDNA encoding human Rab3D whose expression is upregulated with myeloid differentiation.
• Biochim Biophys Acta. 1999; 1444: 283-90
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• To identify genes expressed in myeloid differentiation, we isolated a cDNA fragment by differential display using RNA prepared from HT93A cells, a human cell line capable of differentiating into neutrophil and eosinophil lineages in response to retinoic acid (RA). Evaluation of the full-length clone isolated from an HT93A cDNA library showed that it encoded a 24 kDa protein comprised of several domains conserved in the Ras superfamily. Comparison of the deduced amino acid sequence of this clone with Rab proteins revealed that it had highest homology to a small GTP-binding protein, murine Rab3D. The mRNA expression of human Rab3D was upregulated in the course of myeloid differentiation, and it was preferentially expressed in granulocytes. These results suggest that human Rab3D may play a specific role in granulocytes, for example in exocytosis of neutrophil-specific granules or in degranulation of both eosinophils and basophils.

• Flink IL, Blitz I, Morkin E
• Characterization of cellular nucleic acid binding protein from Xenopus laevis: expression in all three germ layers during early development.
• Dev Dyn. 1998; 211: 123-30
• Display abstract

• The Xenopus CNBP homologue (XCNBP) has been cloned from stage 14 neurula. XCNBP encodes a 18.4-kDa protein containing seven highly conserved zinc finger (Zn-finger) repeats (CX2CX4HX4CX2), with sequence similarity to human, mouse, rat, and yeast CNBP. A unique feature of XCNBP is that it contains a 10 amino acid (aa) deletion in the linker region between Zn-fingers 1 and 2, immediately downstream from an alternatively spliced exon of human CNBP isoforms. A similar deletion is found in mouse and yeast CNBP proteins. The deleted region lacks potential PEST and casein kinase II phosphorylation sites. Because CNBP proteins from a variety of species contain deletions in a similar region, these results suggest that the pattern of alternative processing of CNBP isoforms is highly conserved among metazoa and unicellular eukaryotes. XCNBP RNA is initially maternally derived and is widely expressed throughout early development at the gastrula, neurula, and tailbud stages. At the early gastrula stage, XCNBP is expressed in ectodermal, endodermal, and mesodermal germ layers. Previous data have demonstrated the presence of CNBP in the cytoplasm and nucleus. The interactions of CNBP with single-stranded DNA and RNA suggest that CNBP may serve dual functions in transcriptional and translational regulation in a wide variety of tissues during development.

• Huang HW, Tsoi SC, Sun YH, Li SS
• Identification and characterization of the SMT3 cDNA and gene encoding ubiquitin-like protein from Drosophila melanogaster.
• Biochem Mol Biol Int. 1998; 46: 775-85
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• A SMT3 cDNA encoding ubiquitin-like protein from Drosophila melanogaster was isolated and sequenced. Drosophila SMT3 genomic DNA was amplified by polymerase chain reaction, and its nucleotide sequence was found to be identical to that of the cDNA, indicating the absence of intron in its protein coding region. The sequence of 90 amino acids of Drosophila SMT3 exhibited 55%, 73%, 70% and 52% identity to yeast SMT3, human SMT3A, SMT3B and SMT3C protein.

• Isnard P, Depetris D, Mattei MG, Ferrier P, Djabali M
• cDNA cloning, expression and chromosomal localization of the murine AF-4 gene involved in human leukemia.
• Mamm Genome. 1998; 9: 1065-8

• Vujic M, Rajic T, Goodfellow PN, Stevanovic M
• cDNA characterization and high resolution mapping of the human SOX20 gene.
• Mamm Genome. 1998; 9: 1059-61

• Doi A, Shiosaka T, Takaoka Y, Yanagisawa K, Fujita S
• Molecular cloning of the cDNA encoding A + U-rich element RNA binding factor.
• Biochim Biophys Acta. 1998; 1396: 51-6
• Display abstract

• Using the differential display method, a new cDNA clone, termed laAUF1, encoding the human A + U-rich RNA-binding motif was isolated and sequenced. Analysis of the protein sequence of laAUF1 indicates 73% homology between the deduced polypeptide sequences of laAUF1 and AUF1 in the region encoding a consensus motif for two non-identical RNA recognition motifs (RRMs) and Gln-rich motif. We suggest that the similar affinities of laAUF1 and AUF1 for particular A + U-rich elements (ARE) sequences are related to their potencies as mRNA destabilizers.

• Pahl PM, Hodges YK, Meltesen L, Perryman MB, Horwitz KB, Horwitz LD
• ZNF207, a ubiquitously expressed zinc finger gene on chromosome 6p21.3.
• Genomics. 1998; 53: 410-2

• Seki N et al.
• Isolation, tissue expression, and chromosomal assignment of a novel human gene which encodes a protein with RING finger motif.
• J Hum Genet. 1998; 43: 272-4
• Display abstract

• We identified a novel gene encoding a RING finger (C3HC4-type zinc finger) protein from a human neuroblastoma full-length enriched cDNA library. This cDNA clone consists of 1919 nucleotides with an open reading frame of a 485-amino acid protein. From reverse transcription (RT)-polymerase chain reaction (PCR) analysis, the messenger RNA was ubiquitously expressed in various human adult tissues. The chromosomal location of the gene was determined on the chromosome 6p21.3 region by PCR-based analyses with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel.

• Ogawa M, Hiraoka Y, Taniguchi K, Aiso S
• Cloning and expression of a human/mouse Polycomb group gene, ENX-2/Enx-2.
• Biochim Biophys Acta. 1998; 1395: 151-8
• Display abstract

• The Drosophila Polycomb group (Pc-G) genes encode transcriptional factors involved in development. Little is known about members of the vertebrate Pc-G genes. In this study, we have isolated a cDNA encoding a human Pc-G protein and the mouse equivalent. The human and mouse genes, which were named ENX-2 and Enx-2, encode 702 and 750 amino acids, respectively. ENX-2/Enx-2 protein exhibits a high homology (53-55% identity) to Drosophila Enhancer of zeste [E(z)] protein belonging to the Pc-G. The expression of Enx-2 was observed in mouse kidney, adrenal gland, testis and brain at high levels by Northern blot analysis. A cell line of mouse neuroblastoma, Neuro-2a, also expresses Enx-2 mRNA and its level is elevated by induction of neuronal differentiation of the cell.

• Ishino T, Ohtsuki S, Homma Ki, Natori S
• cDNA cloning of mouse prolyl endopeptidase and its involvement in DNA synthesis by Swiss 3T3 cells.
• J Biochem. 1998; 123: 540-5
• Display abstract

• A cDNA for mouse prolyl endopeptidase (PEP) was cloned and its nucleotide sequence determined. The overall amino acid sequence identity between mouse and other mammalian PEPs was about 96%. A specific inhibitor of PEP, N-benzyloxycarbonyl-thioprolyl-thioprolinal- dimethylacetal (ZTTA), inhibited DNA synthesis by Swiss 3T3 cells. Mouse PEP was shown to be localized partly in restricted nuclear regions. These results suggest that PEP participates in mammalian DNA synthesis.

• Biard DS, Saintigny Y, Maratrat M, Paris F, Martin M, Angulo JF
• Enhanced expression of the Kin17 protein immediately after low doses of ionizing radiation.
• Radiat Res. 1997; 147: 442-50
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• Kin17 is a mammalian nuclear protein sharing a slight sequence homology with the bacterial RecA protein. Kin17 has a zinc-finger motif and binds efficiently to curved DNA, a genomic topology associated with illegitimate recombination junctions. We investigated the relationship between the level of Kin17 protein and genomic alteration due to either impaired wild-type p53 functions or exposure to gamma rays. We used BP cells, a rodent epithelial cell system. The cell lines used were syngeneic and harbored wild-type or mutant p53 alleles and exhibited different sensitivities to gamma irradiation. In radioresistant cells (wild-type p53 genotype), the level of Kin17 protein peaked 30 min after a low dose of radiation (2 Gy), whereas maximum accumulation of p53 protein was observed 3 h postirradiation. Radiosensitive cells carrying the same mutation in both alleles of the p53 gene showed elevated basal levels of both Kin17 and p53 proteins and failed to accumulate Kin17 and p53 proteins after exposure to ionizing radiation. These cells exhibited enhanced cell death by apoptosis after gamma irradiation. Our results indicate that Kin17 protein accumulated immediately after DNA damage in cells carrying a wild-type p53 genotype, and that levels of constitutive Kin17 protein increased in highly proliferating tumorigenic cells when wild-type p53 functions were abrogated.

• Ishikawa S et al.
• Isolation and mapping of a human zinc finger gene (ZNF188) homologous to ZNF187, a serum-response-element binding protein.
• Cytogenet Cell Genet. 1997; 77: 185-9
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• From a human pancreas cDNA library we isolated and characterized a novel zinc finger gene encoding a protein homologous to ZNF187, a serum response element-binding protein. The full-length cDNA contained an open reading frame of 1,686 nucleotides encoding a predicted 562-amino-acid peptide that included an ATP-GTP binding site and seven C2H2 zinc finger domains. The consensus sequence of the C2H2 domains (CX2CX3FX5LX2HX3H) is common in the SRE-binding region present in Drosophila Kruppel proteins. An alternatively spliced form of the transcript found in the cDNA library lacked both the ATP-GTP binding site and any C2H2 zinc finger domains. We localized this gene (ZNF188) to chromosome band 7q22.1-->q22.3 by fluorescence in situ hybridization.

• Becker KG et al.
• Molecular cloning and mapping of a novel developmentally regulated human C2H2-type zinc finger.
• Mamm Genome. 1997; 8: 287-9

• Zhu L, Perlaky L, Henning D, Valdez BC
• Cloning and characterization of a new silver-stainable protein SSP29, a member of the LRR family.
• Biochem Mol Biol Int. 1997; 42: 927-35
• Display abstract

• Silver-stainable proteins (SSPs) are aspartic acid-rich nuclear proteins which are silver stained under very specific conditions. Using a degenerate oligodeoxyncleotide probe which codes for acidic amino acid residues, a cDNA for a new SSP, referred to as SSP29, has been isolated. The cDNA-derived amino acid sequence shows SSP29 has a molecular mass of 29 kDa, leucine-rich repeats (LRR) near the NH2-terminal region and acidic clusters at the COOH-terminal portion, indicating that SSP29 is also a member of the LRR subfamily of acidic proteins which have been shown to be involved in antigen-mediated cellular responses, leukemogenesis and differentiation. SSP29 can be stained by Ag-NOR staining. SSP29 is expressed in all human tissues and cell lines tested, localized to nucleoplasm and translocated partially to the nucleoli after heat shock. Its interaction with RNA polymerase I suggests that SSP29 may participate in signal transduction that directs nucleolar activities by regulating ribosomal RNA biosynthesis.

• Hu G, Chung YL, Glover T, Valentine V, Look AT, Fearon ER
• Characterization of human homologs of the Drosophila seven in absentia (sina) gene.
• Genomics. 1997; 46: 103-11
• Display abstract

• Studies of Drosophila photoreceptor development have illustrated the means by which signal transduction events regulate cell fate decisions in a multicellular organization. Development of the R7 photoreceptor is best understood, and its formation is dependent on the seven in absentia (sina) gene. We have characterized two highly conserved human homologs of sina, termed SIAH1 and SIAH2. SIAH1 maps to chromosome 16q12 and encodes a 282-amino-acid protein with 76% amino acid identity to the Drosophila SINA protein. SIAH2 maps to chromosome 3q25 and encodes a 324-amino-acid protein that shares 68% identity with Drosophila SINA and 77% identity with human SIAH1. SIAH1 and SIAH2 were expressed in many normal and neoplastic tissues, and only subtle differences in their expression were noted. However, one of three murine homologs, Siah1B, was strongly induced in fibroblasts undergoing apoptotic cell death. While a previous study suggested that SINA was a nuclear protein, epitope-tagged SINA and SIAH1 proteins were found in the cytoplasm of Drosophila and mammalian cells. Their substantial evolutionary conservation, role in specifying cell fate, and activation in apoptotic cells suggest the SIAH proteins have important roles in vertebrate development. Furthermore, given the role of sina in Drosophila photoreceptor development, SIAH2 is a candidate for the Usher syndrome type 3 gene at chromosome 3q21-q25.

• Amann J, Kidd VJ, Lahti JM
• Characterization of putative human homologues of the yeast chromosome transmission fidelity gene, CHL1.
• J Biol Chem. 1997; 272: 3823-32
• Display abstract

• Helicases are components of numerous protein complexes, including those regulating transcription, translation, DNA replication and repair, splicing, and mitotic chromosome transmission. Helicases unwind double-stranded DNA and RNA homo- and hetero-duplexes. The yeast CHL1 helicase has been linked to maintenance of the high fidelity of chromosome transmission during mitosis. Mutations in this gene result in a 200-fold increase in the rate of aberrant chromosome segregation with a concomitant delay in the cell cycle at G2-M, suggesting that CHL1 is required for the maintenance of proper chromosome transmission. Two highly related human cDNA clones encoding proteins which are homologous to the yeast CHL1 gene product have been isolated. Here we show that these two distinct human CHL1-related mRNAs and proteins (hCHLR1 and hCHLR2) are expressed only in proliferating human cell lines. Quiescent normal human fibroblasts stimulated to re-enter the cell cycle by addition of serum begin to express the CHL1-related proteins as the cells enter S phase, concomitant with the expression of proliferating cell nuclear antigen. Furthermore, expression of the CHL1-related mRNAs is lost when human K562 cells cease to proliferate and terminally differentiate in response to phorbol ester treatments. Human hCHLR expression is not extinguished during hemin-induced differentiation of the same cell line, which produces erythrocyte-like cells that continue to proliferate. These experiments are consistent with the requirement of this putative helicase during either S or G2-M phase but not G1. In vitro transcribed and translated hCHLR1 protein binds to both single- and double-stranded DNA, supporting the possibility that these proteins are DNA helicases. Finally, affinity-purified hCHLR1 antisera was used to demonstrate the localization of the hCHLR proteins to the nucleolus by indirect immunofluorescence as well as by cell fractionation.

• Fyrberg C, Becker J, Barthmaier P, Mahaffey J, Fyrberg E
• A Drosophila muscle-specific gene related to the mouse quaking locus.
• Gene. 1997; 197: 315-23
• Display abstract

• We have characterized a novel muscle-specific gene of Drosophila melanogaster, defined by enhancer trap strain 24B of Brand and Perrimon (1993). We show that transcripts of the gene accumulate within presumptive mesoderm and persist within developing muscles, strongly suggesting that the encoded protein is involved in muscle cell determination and differentiation. cDNA sequences reveal that the Drosophila protein is similar to quaking (64% identity over 210 amino acids), a protein essential for mouse embryogenesis, and gld-1 (53% identity over 162 amino acids) a germ-line-specific tumor suppressing protein of the nematode, Caenorhabditis elegans. We demonstrate that the Drosophila gene resides within the 93F chromosome subdivision, and describe its physical map. Finally, we have used the gene, which we have named quaking-related 93F (qkr93F), to identify a family of closely related KH domains.

• Izumoto Y, Kuroda T, Harada H, Kishimoto T, Nakamura H
• Hepatoma-derived growth factor belongs to a gene family in mice showing significant homology in the amino terminus.
• Biochem Biophys Res Commun. 1997; 238: 26-32
• Display abstract

• Hepatoma-derived growth factor (HDGF) is an acidic polypeptide with mitogenic activity for fibroblasts performed outside the cells despite the presence of a putative nuclear localization signal (NLS). We have now cloned three related mouse cDNAs: one for a mouse homologue of human HDGF and two for additional HDGF-related proteins provisionally designated HDGF-related proteins 1 and 2 (HRP-1 and -2). Their deduced sequences have revealed that HDGF belongs to a new gene family with a highly conserved 98-amino-acid sequence at the amino terminus (hath region, for homologous to the amino terminus of HDGF). HRP-1 and HRP-2 proteins are 46 and 432 amino acids longer than mouse HDGF, respectively, and have no conserved amino acid sequence other than the hath region. HRP-1 is a highly acidic protein (26% acidic) and also has a putative NLS. HRP-2 protein carries a mixed charge cluster, a sharp switch of positive-to negative-charge residues, which is often found in some nuclear proteins. Northern blotting shows that mouse HDGF and HRP-2 are expressed predominantly in testis and skeletal muscle, to intermediate extents in heart, brain, lung, liver, and kidney, and to a minimal extent in spleen. HRP-1 is expressed specifically in testis. These findings suggest that the HDGF gene family might play a new role in the nucleus especially in testis.

• Gessler M, Klamt B, Tsaoussidou S, Ellis JA, Luzio JP
• The gene encoding the GPI-anchored membrane protein p137GPI (M11S1) maps to human chromosome 11p13 and is highly conserved in the mouse.
• Genomics. 1996; 32: 169-70

• Chiang PW et al.
• Isolation and characterization of the human and mouse homologues (SUPT4H and Supt4h) of the yeast SPT4 gene.
• Genomics. 1996; 34: 368-75
• Display abstract

• To study gene regulation mediated by chromatin in mammals, we isolated the human (SUPT4H) and murine (Supt4h) counterparts of the yeast gene encoding SPT4; the product of this gene presumably interacts with the products of the mammalian homologues (which we have also cloned) of yeast SPT5 and SPT6, thereby modulating chromatin formation and activity. We isolated two different sized human SUPT4H cDNA clones (1464 and 728 nt) and one murine Supt4h (688 nt) cDNA clone; all three encode the same 117-amino-acid protein with conservation of the zinc finger motif found in SPT4. Conservation of this zinc finger motif from yeast to mouse and human implies functional importance. Although the overall sequence homology at the DNA level between the human 728-nt transcript and the murine 688-nt transcript is only 78.4%, the DNA sequence homology is 97.7% within the coding region. At the protein level, the amino acid sequences of the translated murine Supt4h and the human SUPT4H gene products are identical. The likely functional copy of SUPT4H, which has at least two introns, maps to human chromosome 17, with candidate intronless pseudogenes on chromosomes 2, 12, and 20. Buttressing the hypothesis that this is a gene required constitutively, both the human SUPT4H transcripts and the murine Supt4h transcript are expressed widely, although not at equal levels (e.g., such as most histones), in all fetal and adult tissues that we examined.

• Jain MK et al.
• Molecular cloning and characterization of SmLIM, a developmentally regulated LIM protein preferentially expressed in aortic smooth muscle cells.
• J Biol Chem. 1996; 271: 10194-9
• Display abstract

• Differentiated, quiescent vascular smooth muscle cells assume a dedifferentiated, proliferative phenotype in response to injury, one of the hallmarks of arteriosclerosis. Members of the LIM family of zinc-finger proteins are important in the differentiation of various cells including striated muscle. We describe here the molecular cloning and characterization of a developmentally regulated smooth muscle LIM protein, SmLIM, that is expressed preferentially in the rat aorta. This 194-amino acid protein has two LIM domains, and comparisons of rat SmLIM with its mouse and human homologues reveal high levels of amino acid sequence conservation (100 and 99%, respectively). SmLIM is a nuclear protein and maps to human chromosome 3. SmLIM mRNA expression was high in aorta but not in striated muscle and low in other smooth muscle tissues such as intestine and uterus. In contrast with arterial tissue, SmLIM mRNA was barely detectable in venous tissue. The presence of SmLIM expression within aortic smooth muscle cells was confirmed by in situ hybridization. In vitro, SmLIM mRNA levels decreased by 80% in response to platelet-derived growth factor-BB in rat aortic smooth muscle cells. In vivo, SmLIM mRNA decreased by 60% in response to vessel wall injury during periods of maximal smooth muscle cell proliferation. The down-regulation of SmLIM by phenotypic change in vascular smooth muscle cells suggests that it may be involved in their growth and differentiation.

• Hamada T et al.
• Isolation and characterization of a novel secretory protein, stromal cell-derived factor-2 (SDF-2) using the signal sequence trap method.
• Gene. 1996; 176: 211-4
• Display abstract

• With use of the signal sequence trap method, we isolated a cDNA encoding a novel secretory protein, SDF-2, from the mouse stromal cell line, ST2. The human homologue of SDF-2 was also isolated. The amino acid (aa) sequences deduced from both the clones were conserved more than 92%. The chromosomal localization of the human SDF-2 gene was mapped to 17q11.2. The aa sequence of SDF-2 shows similarity to those of yeast dolichyl phosphate-D-mannose:protein mannosyltransferases, Pmt1p [Strahl-Bolsinger et al. (1993) Proc. Natl. Acad. Sci. USA 90, 8164-8168] and Pmt2p [Lussier et al. (1995) J. Biol. Chem. 270, 2770-2775], whose activities have not been detected in higher eukaryotes.

• Susens U, Borgmeyer U
• Characterization of the human germ cell nuclear factor gene.
• Biochim Biophys Acta. 1996; 1309: 179-82
• Display abstract

• A cDNA clone encoding the germ cell nuclear factor, GCNF, a member of the nuclear receptor superfamily has been isolated from the human embryonal carcinoma cell line NT2/D1. Sequencing of this clone reveals an open reading frame encoding a 476 amino acid protein. A comparison of the amino acid sequence of the human GCNF with its mouse homologue shows only six amino acid exchanges in the whole protein and a deletion in the amino-terminal region. Northern blot analysis demonstrates that the expression in the testis is conserved.

• Tranque P, Crossin KL, Cirelli C, Edelman GM, Mauro VP
• Identification and characterization of a RING zinc finger gene (C-RZF) expressed in chicken embryo cells.
• Proc Natl Acad Sci U S A. 1996; 93: 3105-9
• Display abstract

• To identify changes in gene expression that occur in chicken embryo brain (CEB) cells as a consequence of their binding to the extracellular matrix molecule cytotactin/tenascin (CT/TN), a subtractive hybridization cloning strategy was employed. One of the cDNA clones identified was predicted to encode 381 amino acids and although it did not resemble any known sequences in the nucleic acid or protein data bases, it did contain the sequence motif for the cysteine-rich C3HC4 type of zinc finger, also known as a RING-finger. This sequence was therefore designated the chicken-RING zinc finger (C-RZF). In addition to the RING-finger, the C-RZF sequence also contained motifs for a leucine zipper, a nuclear localization signal, and a stretch of acidic amino acids similar to the activation domains of some transcription factors. Southern analysis suggested that C-RZF is encoded by a single gene. Northern and in situ hybridization analyses of E8 chicken embryo tissues indicated that expression of the C-RZF gene was restricted primarily to brain and heart. Western analysis of the nuclear and cytoplasmic fractions of chicken embryo heart cells and immunofluorescent staining of chicken embryo cardiocytes with anti-C-RZF antibodies demonstrated that the C-RZF protein was present in the nucleus. The data suggest that we have identified another member of the RING-finger family of proteins whose expression in CEB cells may be affected by CT/TN and whose nuclear localization and sequence motifs predict a DNA-binding function in the nucleus.

• Taylor-Harris P, Swift S, Ashworth A
• Zfyl encodes a nuclear sequence-specific DNA binding protein.
• FEBS Lett. 1995; 360: 315-9
• Display abstract

• Zfyl is a mouse Y chromosomal gene encoding a zinc finger protein which is thought to have some function during spermatogenesis. Here we show that, when introduced into tissue culture cells, Zfyl is targeted to the nucleus. Two independent signals are present within the protein for nuclear localization. This nuclear Zfyl protein is able to bind strongly to DNA-cellulose and, using site-selection assays, we have identified specific Zfyl DNA binding sites. Taken together these results suggest that Zfyl is a nuclear-located sequence-specific DNA binding protein which functions during spermatogenesis.

• Collazo-Garcia N, Scherer P, Aplan PD
• Cloning and characterization of a murine SIL gene.
• Genomics. 1995; 30: 506-13
• Display abstract

• The human SIL gene is disrupted by a site-specific interstitial deletion in 25% of children with T-cell acute lymphoblastic leukemia. Since transcriptionally active genes are prone to recombination events, the recurrent nature of this lesion suggests that the SIL gene product is transcriptionally active in the cell type that undergoes this interstitial deletion and that the SIL gene product may play a role in normal lymphoid development. To facilitate studies of SIL gene function, we have cloned and characterized a murine SIL gene. The predicted murine SIL protein is 75% identical to the human gene, with good homology throughout the open reading frame. An in vitro translated SIL cDNA generated a protein slightly larger than the predicted 139-kDa protein. Although a prior report detected SIL mRNA expression exclusively in hematopoietic tissues, a sensitive RT-PCR assay demonstrated SIL expression to be ubiquitous, detectable in all tissues examined. Since the RT-PCR assay suggested that SIL mRNA expression was higher in rapidly proliferating tissues, we assayed SIL mRNA expression using a murine erythroleukemia model of terminal differentiation and found it to be dramatically decreased in conjunction with terminal differentiation. These studies demonstrate that the human SIL gene product is quite well conserved in rodents and suggest that the SIL gene product may play a role in cell proliferation.

• Tuggle CK, Schmitz CB, Wang L, Rothschild MF
• Cloning and mapping of porcine OTF1 extends a synteny group conserved on SSC 4 and HSA 1.
• Mamm Genome. 1995; 6: 673-6

• Shibahara K, Asano M, Ishida Y, Aoki T, Koike T, Honjo T
• Isolation of a novel mouse gene MA-3 that is induced upon programmed cell death.
• Gene. 1995; 166: 297-301
• Display abstract

• Typical programmed cell death requires de novo macromolecular synthesis and shares common morphological changes referred to as apoptosis. To elucidate the molecular mechanism of apoptosis, we isolated cDNA clones that are induced in various types of apoptosis by the differential display method. Among such clones, the MA-3 mRNA was induced in all apoptosis-inducible cell lines tested so far, including thymocytes, T cells, B cells and pheochromocytoma. The nucleotide sequence of the MA-3 cDNA predicted an amino acid (aa) sequence of 469 aa, which did not reveal significant similarity to any known proteins and functional aa motifs in databases. The MA-3 mRNA was strongly expressed in the thymus although small amounts of the MA-3 mRNA were ubiquitously expressed in mouse adult tissues. The MA-3 gene was highly conserved during evolution and cross-hybridization bands were found not only in vertebrates but also in Drosophila melanogaster.

• Brinkmann U, Brinkmann E, Gallo M, Pastan I
• Cloning and characterization of a cellular apoptosis susceptibility gene, the human homologue to the yeast chromosome segregation gene CSE1.
• Proc Natl Acad Sci U S A. 1995; 92: 10427-31
• Display abstract

• We recently isolated human cDNA fragments that render MCF-7 breast cancer cells resistant to cell death caused by Pseudomonas exotoxin, Pseudomonas exotoxin-derived immunotoxins, diphtheria toxin, and tumor necrosis factor. We report here that one of these fragments is an antisense fragment of a gene homologous to the essential yeast chromosome segregation gene CSE1. Cloning and analysis of the full-length cDNA of the human CSE1 homologue, which we name CAS for cellular apoptosis susceptibility gene, reveals a protein coding region with similar length (971 amino acids for CAS, 960 amino acids for CSE1) and 59% overall protein homology to the yeast CSE1 protein. The conservation of this gene indicates it has an important function in human cells consistent with the essential role of CSE1 in yeast. CAS is highly expressed in human tumor cell lines and in human testis and fetal liver, tissues that contain actively dividing cells. Furthermore, CAS expression increases when resting human fibroblasts are induced to proliferate and decreases when they are growth-arrested. Thus, CAS appears to play an important role in both toxin and tumor necrosis factor-mediated cell death, as well as in cell proliferation.

• Honore B, Leffers H, Madsen P, Celis JE
• Cloning of a cDNA encoding a novel human nuclear phosphoprotein belonging to the WD-40 family.
• Gene. 1994; 151: 291-6
• Display abstract

• We have cloned and expressed in vaccinia virus a cDNA encoding an ubiquitous 501-amino-acid (aa) phosphoprotein that corresponds to protein IEF SSP 9502 (79,400 Da, pI 4.5) in the master 2-D-gel keratinocyte protein database [Celis et al., Electrophoresis 14 (1993) 1091-1198]. The deduced aa sequence contains 9 Trp residues, some of which are localized in repeats and that characterise the protein as a member of the WD-40 family, a group of proteins having 40-aa repeats containing Trp and Asp [Duronio et al., Proteins 13 (1992) 41-56; Van der Voorn and Ploegh, FEBS Lett. 307 (1992) 131-134]. The protein contains a nuclear targeting signal (KKKGK), and fractionation of transformed human amnion cells (AMA) in karyoplasts and cytoplasts confirmed that it is predominantly localized in the nucleus. Database searching indicated that IEF SSP 9502 is a putative human homologue of the Saccharomyces cerevisiae periodic Trp protein, PWP1, a polypeptide that may play a regulatory role in cell growth and/or transcription.

• Shao W, Pratt K
• Cloning genes for DNA binding proteins: dot-blotting to optimize cDNA expression library screening.
• Anal Biochem. 1994; 218: 465-8

• Shibanuma M, Mashimo J, Kuroki T, Nose K
• Characterization of the TGF beta 1-inducible hic-5 gene that encodes a putative novel zinc finger protein and its possible involvement in cellular senescence.
• J Biol Chem. 1994; 269: 26767-74
• Display abstract

• Transforming growth factor (TGF) beta 1 is a potent cytokine that inhibits the growth of several types of cells. Our earlier study suggested that the mouse osteoblastic cell line, MC3T3-E1, was sensitive to growth inhibition by TGF beta 1 and that this effect was partly mediated by H2O2. To identify the molecules that participate in the negative regulation of growth by these stimuli, we carried out differential screening of cDNA libraries and isolated a set of genes induced by TGF beta 1. Among the clones isolated, one originally named tsc-5 was found to be induced by H2O2 as well as TGF beta 1. Analysis of this cDNA renamed hic (hydrogen peroxide-inducible clone)-5 suggested that Hic-5 protein has four LIM motifs, each of which contained two (or one) putative zinc fingers. The expression of hic-5 mRNA was repressed in Ki-ras-transformed mouse fibroblasts and in several cell lines established from human tumor. On the other hand, its expression was augmented in the in vitro senescent process of human diploid fibroblasts. Among the mouse organs examined, hic-5 was highly expressed in the lung and spleen. Finally, a colony formation assay using an hic-5 expression vector driven by the cytomegalovirus promoter suggested that hic-5 overexpression had a cytostatic effect on cellular growth, depending upon the cell type. Although the relationship between hic-5 function and the signal transduction pathway of TGF beta 1 remains unresolved, these results implied that hic-5 has some role in the growth-inhibitory pathway associated with in vitro senescence, and that down-regulation of hic-5 contributes to tumorigenesis.

• Schroeder WT, Stewart-Galetka S, Mandavilli S, Parry DA, Goldsmith L, Duvic M
• Cloning and characterization of a novel epidermal cell surface antigen (ESA).
• J Biol Chem. 1994; 269: 19983-91
• Display abstract

• We report here the isolation and characterization of a cDNA that encodes a novel extracellular epidermal molecule, epidermal surface antigen (ESA), which is thought to play a role in intercellular epidermal adhesion. Sequence analysis reveals that the 379 amino acid ESA has a molecular mass of about 41.7 kDa and an alpha-helix-rich secondary conformation. Much of this also has an heptad substructure, consistent with the formation of several bundles of alpha-helices in a compact globular structure. The ESA protein appears to consist of an NH2-terminal hydrophobic region with mixed alpha and beta structure followed by a more hydrophilic COOH-terminal region which is very rich in alpha-helix. The 2.5-kilobase ESA mRNA is expressed in cultured keratinocytes, melanocytes, fibroblasts, carcinoma, and melanoma cell lines. The ESA gene is conserved in all mammalian species examined and has been localized to human chromosome 17 (M17S1) in the same region as the gene for von Recklinghausen neurofibromatosis. The high level of expression of the ESA mRNA in human skin and in cultured cells derived from the epidermis, the appearance of ESA protein early in human development, and conservation of the ESA gene throughout mammalian evolution suggest that the novel ESA protein plays a vital role in epidermal structure and maintenance.

• Murnane JP, Zhu Y, Young BR, Christman MF
• Expression of the candidate A-T gene ATDC is not detectable in a human cell line with a normal response to ionizing radiation.
• Int J Radiat Biol. 1994; 66: 7784-7784
• Display abstract

• Nucleotide sequence analysis of a candidate gene for A-T group D (ATDC) demonstrated that it is related to a group of proteins that contain both zinc finger and leucine zipper motifs. The presence of a leucine zipper suggested that this protein might form homodimers, and this was confirmed by means of the two-hybrid system in yeast. The activity of some proteins that form homodimers can be effectively eliminated by overexpression of inactive forms of the protein that bind to the wild-type protein to create a dominant negative phenotype. An ATDC cDNA containing a 37 amino acid deletion in the zinc finger region (ATDC delta) was therefore transfected into colorectal carcinoma human tumour cells (RKO) to determine whether its expression would produce a response to radiation similar to that seen in A-T cells. RKO cells have been shown to have normal radiosensitivity and cell cycle regulation and, therefore, seemed ideal for this study. Despite the fact that the A-T gene has been found to be important in the radiation damage response, no ATDC mRNA transcripts were detectable in the RKO cell line. In addition, the RKO subclones expressing the ATDC delta mRNA showed no change in radiosensitivity or cell cycle regulation. These results do not support the conclusion that ATDC is an A-T gene, and suggest that the ATDC protein acts indirectly to suppress radiosensitivity in A-T cells.

• Bouchard ML, Cote S
• The Drosophila melanogaster developmental gene g1 encodes a variant zinc-finger-motif protein.
• Gene. 1993; 125: 205-9
• Display abstract

• In Drosophila melanogaster, the mechanisms involved in the pattern formation of complex internal organs are still largely unknown. However, the identity of the molecular determinants that control the development of these specific tissues is emerging from the combined use of genetic and molecular approaches. We have cloned a gene that is expressed in the mesoderm, one of the fundamental embryonic germ layers which gives rise to internal structures, such as the musculature. Here, we describe the molecular characterization of this gene, designated as g1. The nucleotide (nt) sequence of its cDNA shows an open reading frame of 852 nt, which encodes a 32-kDa protein with two putative zinc fingers, and a serine/glutamine/proline-rich region. These features indicate a functional role for g1, which remains to be elucidated, in regulating gene expression during mesoderm formation.

• Su L, Hershberger RJ, Weissman IL
• LYAR, a novel nucleolar protein with zinc finger DNA-binding motifs, is involved in cell growth regulation.
• Genes Dev. 1993; 7: 735-48
• Display abstract

• A cDNA encoding a novel zinc finger protein has been isolated from a mouse T-cell leukemia line on the basis of its expression of a Ly-1 epitope in a lambda gt11 library. The putative gene was mapped on mouse chromosome 1, closely linked to Idh-1, but not linked to the Ly-1 (CD5) gene. The cDNA is therefore named Ly-1 antibody reactive clone (LYAR). The putative polypeptide encoded by the cDNA consists of 388 amino acids with a zinc finger motif and three copies of nuclear localization signals. Antibodies raised against a LYAR fusion protein reacted with a protein of 45 kD on Western blots and by immunoprecipitation. Immunolocalization indicated that LYAR was present predominantly in the nucleoli. The LYAR mRNA was not detected in brain, thymus, bone marrow, liver, heart, and muscle. Low levels of LYAR mRNA were detected in kidney and spleen. However, the LYAR gene was expressed at very high levels in immature spermatocytes in testis. The LYAR mRNA is present at high levels in early embryos and preferentially in fetal liver and fetal thymus. A number of B- and T-cell leukemic lines expressed LYAR at high levels, although it was not detectable in bone marrow and thymus. During radiation-induced T-cell leukemogenesis, high levels of LYAR were expressed in preleukemic thymocytes and in acute T leukemia cells. Fibroblast cells overexpressing the LYAR cDNA from a retrovirus vector, though not phenotypically transformed in vitro, had increased ability to form tumors in nu/nu mice. Therefore, LYAR may function as a novel nucleolar oncoprotein to regulate cell growth.

• Abeliovich H, Tzfati Y, Shlomai J
• A trypanosomal CCHC-type zinc finger protein which binds the conserved universal sequence of kinetoplast DNA minicircles: isolation and analysis of the complete cDNA from Crithidia fasciculata.
• Mol Cell Biol. 1993; 13: 7766-73
• Display abstract

• Replication of the kinetoplast DNA minicircle light strand initiates at a highly conserved 12-nucleotide sequence, termed the universal minicircle sequence. A Crithidia fasciculata single-stranded DNA-binding protein interacts specifically with the guanine-rich heavy strand of this origin-associated sequence (Y. Tzfati, H. Abeliovich, I. Kapeller, and J. Shlomai, Proc. Natl. Acad. Sci. USA 89:6891-6895, 1992). Using the universal minicircle sequence heavy-strand probe to screen a C. fasciculata cDNA expression library, we have isolated two overlapping cDNA clones encoding the trypanosomatid universal minicircle sequence-binding protein. The complete cDNA sequence defines an open reading frame encoding a 116-amino-acid polypeptide chain consisting of five repetitions of a CCHC zinc finger motif. A significant similarity is found between this universal minicircle sequence-binding protein and two other single-stranded DNA-binding proteins identified in humans and in Leishmania major. All three proteins bind specifically to single-stranded guanine-rich DNA ligands. Partial amino acid sequence of the endogenous protein, purified to homogeneity from C. fasciculata, was identical to that deduced from the cDNA nucleotide sequence. DNA-binding characteristics of the cDNA-encoded fusion protein expressed in bacteria were identical to those of the endogenous C. fasciculata protein. Hybridization analyses reveal that the gene encoding the minicircle origin-binding protein is nuclear and may occur in the C. fasciculata chromosome as a cluster of several structural genes.

• Andreazzoli M, De Lucchini S, Costa M, Barsacchi G
• RNA binding properties and evolutionary conservation of the Xenopus multifinger protein Xfin.
• Nucleic Acids Res. 1993; 21: 4218-25
• Display abstract

• Xfin is a Xenopus zinc finger protein which is expressed in the cytoplasm of the oocyte and throughout embryogenesis, as well as in the cytoplasm of some specific and highly differentiated cell types (De Lucchini et al., Mech. Dev. 36, 31-40, 1991). In this paper we present a characterization of some structural features of the protein and of its nucleic acid binding properties. We found that Xfin is a phosphoprotein, is present in the soluble fraction of the cytoplasm, and is actively phosphorylated in cytosolic extracts. Several putative phosphorylation sites are present in the cDNA-derived protein sequence, mostly located at specific positions within the Zn-fingers. In an in vitro assay a fusion protein containing part of the finger region of Xfin exhibits specific binding to a poly (G) RNA homopolymer, while it does not bind DNA. The RNA binding activity of the protein is significantly enhanced by phosphorylation. A putative Xfin homolog, which appears to be evolutionarily conserved with regard to size, cytoplasmic expression and antigenic specificity, is present in representatives of five Vertebrate classes. Taken together, these results may suggest that, by virtue of its RNA binding activity modulated through phosphorylation, Xfin could serve some evolutionarily conserved function in post-transcriptional regulation processes.

• Sailer A, MacDonald NJ, Weissmann C
• Cloning and sequencing of the murine homologue of the human splicing factor U2AF65.
• Nucleic Acids Res. 1992; 20: 2374-2374

• Montecucco A, Biamonti G, Savini E, Focher F, Spadari S, Ciarrocchi G
• DNA ligase I gene expression during differentiation and cell proliferation.
• Nucleic Acids Res. 1992; 20: 6209-14
• Display abstract

• We have studied the regulation of mammalian DNA ligase I gene by using a cDNA probe in Northern blot experiments with RNA extracted from several cell types in different growth conditions. DNA ligase I mRNA is detected in all analysed cell systems, regardless of their proliferation state, including mature rat neurons. A significant increase in DNA ligase I mRNA level is observed when cells are induced to proliferate, in agreement with the raise of DNA joining activity found in the same cell systems. The increase parallels the start of DNA synthesis, but the messenger remains at high level beyond the end of the S phase and is detected also in the presence of aphidicolin. A decrease in DNA ligase I mRNA is observed in HL-60 and NIH-3T3 cells after differentiation. The high stability of DNA ligase I mRNA in both resting and proliferating human fibroblasts suggests a cell proliferation dependent rate of transcription. On the other hand the presence of a basal level of DNA ligase I in nondividing cells, strongly suggests an involvement of this enzyme in DNA repair. This conclusion is supported by a threefold increase in DNA ligase I observed 24 h after UV irradiation of human confluent primary fibroblasts.

• Chardin P, Courtois G, Mattei MG, Gisselbrecht S
• The KUP gene, located on human chromosome 14, encodes a protein with two distant zinc fingers.
• Nucleic Acids Res. 1991; 19: 1431-6
• Display abstract

• We have isolated a human cDNA (kup), encoding a new protein with two distantly spaced zinc fingers of the C2H2 type. This gene is highly conserved in mammals and is expressed mainly in hematopoietic cells and testis. Its expression was not higher in the various transformed cells tested than in the normal corresponding tissues. The kup gene is located in region q23-q24 of the long arm of human chromosome 14. The kup protein is 433 a.a. long, has a M.W. close to 50 kD and binds to DNA. Although the structure of the kup protein is unusual, the isolated fingers resemble closely those of the Kruppel family, suggesting that this protein is also a transcription factor. The precise function and DNA motif recognized by the kup protein remain to be determined.

• Fornace AJ Jr et al.
• Mammalian genes coordinately regulated by growth arrest signals and DNA-damaging agents.
• Mol Cell Biol. 1989; 9: 4196-203
• Display abstract

• More than 20 different cDNA clones encoding DNA-damage-inducible transcripts in rodent cells have recently been isolated by hybridization subtraction (A. J. Fornace, Jr., I. Alamo, Jr., and M. C. Hollander, Proc. Natl. Acad. Sci. USA 85:8800-8804, 1988). In most cells, one effect of DNA damage is the transient inhibition of DNA synthesis and cell growth. We now show that five of our clones encode transcripts that are increased by other growth cessation signals: growth arrest by serum reduction, medium depletion, contact inhibition, or a 24-h exposure to hydroxyurea. The genes coding for these transcripts have been designated gadd (growth arrest and DNA damage inducible). Two of the gadd cDNA clones were found to hybridize at high stringency to transcripts from human cells that were induced after growth cessation signals or treatment with DNA-damaging agents, which indicates that these responses have been conserved during mammalian evolution. In contrast to results with growth-arrested cells that still had the capacity to grow after removal of the growth arrest conditions, no induction occurred in HL60 cells when growth arrest was produced by terminal differentiation, indicating that only certain kinds of growth cessation signals induce these genes. All of our experiments suggest that the gadd genes are coordinately regulated: the kinetics of induction for all five transcripts were similar; in addition, overexpression of gadd genes was found in homozygous deletion c14CoS/c14CoS mice that are missing a small portion of chromosome 7, suggesting that a trans-acting factor encoded by a gene in this deleted portion is a negative effector of the gadd genes. The gadd genes may represent part of a novel regulatory pathway involved in the negative control of mammalian cell growth.

• Sukhatme VP et al.
• A zinc finger-encoding gene coregulated with c-fos during growth and differentiation, and after cellular depolarization.
• Cell. 1988; 53: 37-43
• Display abstract

• Egr-1 is an early growth response gene that displays fos-like induction kinetics in fibroblasts, epithelial cells, and lymphocytes following mitogenic stimulation. Sequence analysis of murine Egr-1 cDNA predicts a protein with three DNA binding zinc fingers. The human EGR1 gene maps to chromosome 5 (bands 5q23-31). Egr-1 mRNA increases dramatically during cardiac and neural cell differentiation, and following membrane depolarization both in vitro and in vivo. Thus, Egr-1 and c-fos are often coregulated with strikingly similar kinetics. These results, in conjunction with the Egr-1 primary structure, suggest that Egr-1 may function as a transcriptional regulator in diverse biological processes.

• Celis JE, Madsen P, Nielsen S, Petersen Ratz G, Lauridsen JB, Celis A
• Levels of synthesis of primate-specific nuclear proteins differ between growth-arrested and proliferating cells.
• Exp Cell Res. 1987; 168: 389-401
• Display abstract

• A monoclonal antibody that reacts specifically with the proliferation-sensitive nuclear proteins, isoelectric focusing (IEF) 8Z30 and 8Z31 (molecular weight (MW), 76,000 charge variants, HeLa protein catalogue number) has been characterized. As determined by indirect immunofluorescence, the antibody stains the nucleolus and nucleoplasm of interphase-cultured cells of primate origin, but does not react with cells of other species. Proteins having similar MWs and isoelectric points as the human or monkey (primates) proteins were not observed in cultured cells of the following species: aves, bat, dog, dolphin, goat, hamster, mink, mouse, pisces, potoroo, rabbit and rat. Quantitative two-dimensional (2D) gel electrophoretic analysis of [35S]methionine-labeled proteins synthesized by normal (quiescent, proliferating) and SV40-transformed human MRC-5 fibroblasts revealed significant differences in the levels of synthesis of both IEF 8Z30 and 8Z31. In quiescent cells the main labelled product corresponded to IEF 8Z31 (ratio IEF 8Z31/8Z30, 2.3), while in the transformed cells the major product was IEF 8Z30 (ratio, 0.62). Normal proliferating fibroblasts exhibited similar levels of both proteins (ratio, 1.21). Combined levels of synthesis of both proteins were 1.50 and 1.20 times as high in the transformed cells as in the quiescent and proliferating cells, respectively. Similar results were observed in other pairs of normal and transformed human cells, such as WI38/WI38 SV40 and amnion/AMA. Modulation of the levels of synthesis of these proteins may play a role in cell proliferation.

• Schmidtke J, Krawczak M, Cooper DN
• Human gene cloning: the storm before the lull?
• Nature. 1986; 322: 119-119

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